A hair follicle, a tissue producing hair, is formed in a certain period of the embryonic stage by an interaction between a special mesenchyme and the epidermis called a dermal condensation. Cells constituting such a dermal condensation become a dermal papilla after hair follicle formation is completed. The dermal papilla also deeply participates in the progression of hair cycle which is a cycle of anagen and telogen of hair. In order to induce hair follicle formation by cell transplantation, this interaction between the epidermis and dermal papilla (mesenchyme) should be allowed to take place in the living body. The present inventors have invented a method for growing hair shafts from this hair follicles as a means for allowing hairs to grow on a hairless area on the skin in which mixed epidermal and dermal papilla cells are transplanted in the skin, whereby the human dermal papilla cells induce the epidermal cells into a hair follicles, and hair shafts are allow to grow from this hair follicles, and have already applied for patent (Patent document 1).
Further, it has been reported that dermal papilla cells isolated from rat whiskers and primary cultured can be subcultured for a long period of time by adding a supernatant of conditioned medium of primary culture of rat sole epidermal cells, and the dermal papilla cells subcultured for a long period of time sustain an ability to induce hair follicle formation (Non-patent document 1, Patent document 2). However, the present inventors have shown that the rat whisker-derived dermal papilla cells proliferated by long-term subculture (for about more than 40 passages) using the method described in Patent document 2 sustain an ability to induce hair follicle formation, but an ability to grow hair shafts from the hair follicles whose formation has been induced is reduced and lost (Patent document 1). As a method for solving this problem, the present inventors have made it possible to restore the reduced ability to induce hair growth by adding a given amount of dermal sheath cells of hair follicles to dermal papilla cells whose ability to grow hair shafts has been reduced due to long-term subculture, and to significantly promote the growth of hair emerging from the hair follicles, and have applied for patent (Patent document 1).
It is defined that the dermal sheath is a tissue composed of one or several dermal cell layer(s) (positive to vimentin) surrounding the outermost layer of the hair follicle, and is distributed in the lower one-third or less of the hair follicle, and is composed of smooth muscle-α-actin (α-SMA) positive cells. This dermal sheath is histologically continuous with the dermal papilla at the lowest end of the hair bulb. Further, when the rat whisker hair follicle is cut above and below and hair follicular dermal sheath cells of the hair bulb are transplanted by implantation in the upper part of the hair follicle, the hair bulb including dermal papilla derived from the hair follicular dermal sheath cells is reconstructed and elongation of hair shaft is observed. From these events, it has been indicated that precursor cells of dermal papilla cells are distributed in the hair follicular dermal sheath, and are a supply source of cells to the dermal papilla (Non-patent document 3).
It has been reported that based on these findings, when the hair follicular dermal sheath of a male was cross-transplanted in the skin of a forearm area of a female, the formation of the hair follicle including the dermal papilla derived from the transplanted dermal sheath was induced, and hair was allowed to grow (Non-patent document 4). Further, it has been reported that when primary cultured rat hair follicular dermal sheath cells were allografted in just under the epidermis of a hairless skin area of the rat auricle, the hair follicle formation was induced from the epidermis of the auricle, resulting in hair growth (Non-patent document 5). From these events, hair follicular dermal sheath cells include precursor cells of dermal papilla cells and can be used as a supply source of the dermal papilla cells.
Further, it has been reported that hair follicular dermal sheath cells are promising not only as a supply source of dermal papilla cells, but also as a wound healing material (Non-patent documents 6 and 7, Patent document 3). In a study using a rat, when cultured hair follicular dermal sheath cells derived from a whisker were transplanted in the dorsal area of a nude mouse in combination with cells prepared from the hair follicular outer root sheath, normal skin was formed. Further, when the epidermis formation of bell-type artificial skin composed of human cells and collagen gel was compared between fibroblasts derived from human foreskin and hair follicular dermal sheath cells derived from scalp hair follicles, and as a result, apparently, a thicker epidermal layer was formed when hair follicular dermal sheath cells were used. From this, it can be said that dermal sheath cells have an ability to differentiate not only into the dermal papilla but also into fibroblasts which form good dermis.
As described above, hair follicular dermal sheath cells can be used for improvement of the quality in hair follicle regeneration, and as a supply source of dermal papilla cells and fibroblasts of artificial skin. Further, because the cells have a function of promoting elongation of the hair shafts, it can be said that the cells have an extremely high utility value in the development of pharmaceuticals such as hair growth agents and therapeutic agents for alopecia targeting the hair follicular dermal sheath cells. However, the human hair follicular dermal sheath is a small tissue surrounding the hair follicle with almost a single layer of cells, and the number of obtained cells is extremely small. Further, with a conventional culture method, the proliferation rate of hair follicular dermal sheath cells in primary culture is extremely low. Generally, in the case where the proliferation rate in primary culture is low, the risk of contamination by fungi and bacteria becomes very high, and it becomes difficult to secure a cell number that enables the application thereof to a treatment or development of pharmaceuticals. Accordingly, a culture method in which primary culture with a small amount of hair follicular dermal sheath tissue at a proliferation rate as high as possible is carried out and then, cells which sustain a definite function can be secured in sufficient quantity has been demanded. However, it has been confirmed that the rat hair follicular dermal sheath cells subjected to primary culture have an ability to induce the hair follicle and to grow hair so far, but it has not been confirmed that cells proliferated by subculture using a conventional culture technique have a significant ability to induce the hair follicle and to grow hair or ability to form the hair follicular dermal sheath (Non-patent document 5).
As discussed in the above, by culturing and propagating cells having a specific function while sustaining a definite function, cells having a high utility value can be obtained in a large amount. However, if the differentiation of pluripotent stem cells or precursor cells into cells which are capable of differentiating into a specific cell species and have a high proliferation potential can be controlled by culture, not only a similar result can be obtained, but also targeted cells can be efficiently obtained from a small amount of tissue or cells. On the other hand, dermal sheath cells disappear in the telogen phase in hair cycle, therefore, the existence of precursor cells thereof can be expected. However, there has been no report so far of the existence of the precursor cells or a culture method for the precursor cells.
Generally, in a cell culture method, various growth factors are added to a culture medium and proliferation and differentiation of cells are controlled. It has been reported that dermal sheath cells are precursor cells of dermal papilla cells, and a culture method with the addition of various growth factors has been reported. Among them, there are reports that the addition of fibroblast growth factor 2 (FGF2) or a supernatant of epidermal cell culture is effective in cell proliferation (Non-patent documents 1 and 9). Further, in cultured dermal papilla cells, a subtype of platelet-derived growth factor (PDGF), PDGF-AA does not promote proliferation, but PDGF-BB promotes proliferation in a concentration dependent manner. However, both subtypes of PDGF promote the proliferation of cultured fibroblasts, therefore, it cannot be said that PDGF-BB specifically promotes the proliferation activity of dermal papilla cells. Further, the function of these proliferated cultured dermal papilla cells is not clearly shown. The present inventors have enabled the proliferation of dermal papilla cells that sustain an ability to induce hair follicle formation using the method described in Patent document 2. However, it is difficult to effect culture and proliferation of hair follicular dermal sheath cells using the same method. In the method of Patent document 1, rat whisker-derived hair follicular dermal sheath cells were sufficiently proliferated using a culture medium supplemented with FGF2, and the resulting cultured rat hair follicular dermal sheath cells did not have an ability to induce hair growth alone, but showed a significant ability to grow hair by the addition thereof to dermal papilla cells. However, in human scalp hair-derived hair follicular dermal sheath cells, sufficient cell proliferation cannot be obtained even if a culture medium supplemented with FGF2 is used.
As described above, hair follicular dermal sheath cells are potent cellular materials for such as hair regeneration by cell transplantation, however, there has been no means for well proliferating such hair follicular dermal sheath cells while sustaining their function so far. Further, there has been no report so far of the existence of precursor cells of dermal sheath cells. However, it is apparent that a culture method capable of proliferating newly found dermal sheath precursor cells while sustaining a high proliferation activity and an ability to differentiate into dermal sheath and dermal papilla cells has an extremely high applicability and utility value.    Patent document 1: International Pamphlet for PCT/JP 2004/018421    Patent document 2: JP-A-7-274950    Patent document 3: JP-T-2002-507132    Non-patent document 1: Inamatsu, M. et al., Establishment of rat dermal papilla cell lines that sustain the potency to induce hair follicles from afollicular skin. J. Invest. Dermatol, 111; 767-775, 1998    Non-patent document 2: Weinberg W C et al., Reconstitution of hair follicle development in vivo; determination of follicle formation, hair growth, and hair quality by dermal cells. J. invest. Dermatol., 100, 229-236, 1993    Non-patent document 3; Horne K A and Jahoda C A B, Restoraction of hair growth by surgical implantation of follicular dermal sheath. Development 116, 563-571, 1992    Non-patent document 4: Reynolds A J et al., Trans-gender induction of hair follicles. Nature, 402, 33-34, 1999    Non-patent document 5: McElwee K J et al., Cultured peribulbar dermal sheath cells can induce hair follicle development and contribute to the dermal sheath and dermal papilla. J. invest. Dermatol, 121, 1267-1275, 2003    Non-patent document 6; Jahoda C A B and Reynolds A J, Hair follicle dermal sheath cells: unsung participants in wound healing. Lancet, 358, 1445-1448, 2001    Non-patent document 7: Gharzi A et al., Plasticity of hair follicle dermal cells in wound healing and induction. Exp. Dermatol, 12, 126-136, 2003    Non-patent document 8: Goodman L V and Ledbetter S R, Secretion of stromelysin by cultured dermal papilla cells: differential regulation by growth factors and functional role in mitogen-induced cell proliferation. J. Cellular Physiol, 151, 41-49, 1992    Non-patent document 9: Dawen Yu et al., Expression profiles of tyrosine kinases in cultured follicular papilla cells versus dermal fibroblasts, J. Invest. Dermatol, 123, 283-290, 2004    Non-patent document 10; Jahoda C A B et al., Smooth muscle a-actin is a marker for hair follicle dermis in vivo and in vitro. J. Cell Science, 99, 627-636, 1991    Non-patent document 11: A quantitative study of the differential expression of alpha-smooth muscle actin in cell populations of follicular and non-follicular origin. J. invest. Dermatol. 101, 577-583, 1993    Non-patent document 12: Oliver R F, Histological studies of whisker regeneration in the hooded rat. J. Embryol. exp. Morph., vol. 16, 2, 231-244, October 1966    Non-patent document 13: Oliver R F, Ectopic regeneration of whiskers in the hooded rat from implanted lengths of vibrissa follicle wall. J. Embryol. exp. Morph., vol. 17, 1, 27-34, February 1967